lnternotronal Jounwlfor Parasirology Vol. 15,No. 4pp.353-359. 1985,
Printed in Great Britain. 0
Pergamon Press Ltd. 1985 Australian Society for Pmzsitology
INFLUENCE OF PRIMARY INFECTION ON THE POPULATION DYNAMICS OF NEMATUSPIROIDES DUBIWS AFTER CHALLENGE INFECTIONS IN MICE COLON DOBSON, PBTRUS SITEPU
and PAUL J. BRINDLEY
Department of Parasitology, University of Queensland, St. Lucia, Brisbane, 4067, Australia (Received 18 April 1984; in revised form 25 October 1984) Abstr~et-~~~
C., SITEPU P. and BRINDLEY P. J. 1985. Influence of primary infection on the population dynamics of ~emufos~iro~des dubius after challenge infections in mice. rnternat~onai Journal for ~~rasjtology 15: 3.53-359. Similar proportions of the inoculum of ~emat~piroid~ dub&s larvae reached sexual maturity by 14 days after administration of 50-400 larvae but more adult worms had been expelled by day 63 after infection from those mice infected with 50 vs 400 larvae. There was a significant correlation between time and worm expulsion for all inoculum size groups except for mice given 400 larvae. In mice reinfected with 100 larvae, after termination of primary infections derived from 10 through 400 larvae, more worms from the challenging dose were recovered from mice given greater compared with those given smaller numbers of larvae at primary infection. The N. dubius population size after challenge infection was correlated positively both with number of larvae administered as the primary infection and with the resultant population size during that infection. The serum anti-N. dubius antibody titres after reinfection were higher in mice given 400 compared with those given fewer larvae at primary infection, and the fecundity and female to male sex ratio of the N. dub&s populations decreased in proportion to these antibody titres. Protective immunity against challenge N. dubius infection, in mice which had been drenched free of adult worms established from 400 larvae for 5 down to 1 weeks before reinfection, increased from 45% (1 week) to 80% (5 weeks). There was a negative correlation between the population size of N. dubius during challenge infection and the duration between anthelminti~ treatment and challenge infection.
INDEX KEY WORDS: Nematospirodes dubius; challenge infections; population dynamics; fecundity; immunosuppression; mice. INTRODUCTION REPEATED
of mice with Nematospiroides
dubius larvae stimulates strong immunity to this parasite (Panter, 1969; Bartlett & Ball, 1972; Kerboeuf & Jolivet, 1980) and this phenomenon is modulated by host genotype (Prowse, Mitchell, Ey & Jenkin, 1979; Sitepu & Dobson, 1982). Parenteral administration of viable larvae, adult worms or worm extracts aiso generates varying degrees of immunity to challenge infection (Cypess, 1970, Hitch0 & Thorsen, 1974; Cypess & Zidian, 1975; Hurley, Day & Mitchell, 1980; Mitchell & Munoz, 1983). However, primary infections in mice are characterized by infection of extended duration (Ehrenford, 1954) and by IgCl hypergammaglobulinaemia (Crandall, Crandall & France, 1974; Chapman, Knopf, Anders & Mitchell, 1979). The longevity of this infection can be attributed to the immunosuppressive activity induced by adult N. dub&s populations on the progress of host protective
Correspondence to: Dr C. Dobson, Department of Parasitology, University of Queensland. St. Lucia, Brisbane, 4067, Australia.
responses (Dobson & Cayzer, 1982a, b; Behnke, Hannah & Pritchard, 1983; Cayzer & Dobson, 1983; Sitepu, Dobson & Brindley, in press). The putative advantage for N. dubius releasing immunosuppressive molecules into their environment to enhance their survival is obvious. Indeed, other species of gut dwelling worms may benefit from the immunosuppressive activities of N. dubi~ during concurrent infection (Jenkins 8c Behnke, 1977; Behnke, Wakelin &W&on, 1978). This study investigated the effect of increasingly larger doses of N, dubius larvae on the survival of adult worms during primary infection, on the ability of mice immunized with SO-400 larvae to resist challenge infection, and on the duration of immunosuppression after primary infections had been removed. MATERIALS AND METHODS Mice and Nematospiroides
dubius. Female, B-week-old allogenic Quackenbush (Qf strain mice obtained from the Central Animal Breeding House of the University of Queensland were used in all these experiments. N. dub&s was maintained in Q mice. All the parasites used in these experiments were from the 20th to the 30th generation 353
I.J.P. VOL.15. 1985
COLINDOSSON,PETRUSSITEPUand PAULJ. BRINDLEY
passaged through this host (Dobson & Owen, 1977). Infective larvae were obtained from 7-day-old faecal cultures, concentrated by sedimentation and stored in a shallow layer of water at 4°C in sealed bottles. Larvae were aged for 2 weeks prior to inoculation and the number in each suspension adjusted to give the required dose in 0.2 ml water. The sampling error was < 3%. Mice were infected by stomach intubation. Collection of seru. Blood was taken from mice under ether anaesthesia by cardiac puncture and the serum collected and stored at -20°C. Parasitological techniques. Mice were bled, killed by cervical dislocation, flayed, the peritoneum opened and the intestines stripped from the mesenteries and teased open with forceps on a glass plate under a stereo microscope. The worms were removed, sexed and counted. Mice were drenched free of worms with 20 mg kg-1 levamisole phosphate (Nilverm, ICI, Australia) per OS and the faeces of each drenched mouse were checked and were always free of parasite eggs by 3 days after this treatment. Numbers of N. dubius eggs in the faeces of mice were determined as epg using the McMaster technique (Roberts & O’Sullivan, 1950), and were used to estimate the fecundity of female N. dubius as eggs shed per female worm per day (efd) (Brindley & Dobson, 1982). Antibody titres. Serum anti-N. dubius antibody titres from individual mice were assayed by passive haemagglulination (Ling, 1961) using saline extracts of adult N. dubius worms as antigen (Dobson, 1982). Experimental designs. Experiment 1 tested the effect of N. dubius larval dose on the rate of expulsion of the resultant adult worm populations from mice. Groups of 100 mice were infected with 50, 100, 200 or 400 larvae. Ten other mice were infected with 100 larvae to compare the infectivity of separate cultures of larvae used among the experiments described here because of possible environmental influences (Kerboeuf, 1978). These control mice, monitoring infectivity of the larvae, were killed 3 weeks after infection and adult N. dubius were recovered and counted. Twenty mice from each dose group were killed 2, 3,4, 5 and 9 weeks after infection and the worms recovered, sexed and counted. Experiment 2 examined the effect of the size of a primary N. dubius inoculum on the fate of secondary infections given after chemical termination of the primary infections. Groups of 20 mice were infected with 10, 25, 50, 100, 200 or 400 larvae together with 10 infectivity control mice given 100 larvae. Faecal epg counts were done 19, 20 and 21 days after infection when half the mice and the larval infectivity controls were killed and searched
for parasites; the remainder were drenched free of parasites with anthelmintic and reinfected with 100 N. dubius 4 days later together with 10 mice as larval infectivity controls. Faecal epg counts were done 19, 20 and 21 days after reinfection when the mice were exsanguinated, killed and the parasites recovered, sexed and counted. This experiment was repeated and the two results were pooled. Experiment 3 investigated the loss of residual immunosuppression induced by N. dub&s after termination of primary N. dubius infections. Two hundred and sixty mice were infected with 400 larvae, together with 10 mice given 100 larvae as larval infectivity controls. Twenty test mice plus the mice infected as infectivity controls were killed after 4 weeks of infection. Forty infected mice were drenched free of N. dubius infection with anthelmintic 4 weeks after infection, and at the same time 10 non-infected mice were similarly treated with anthelmintic. Groups of 20 infected mice were killed each week from week 5 to 8, and their parasites recovered. Other groups, each of 20 mice, were drenched free of parasites at week 5, 6, 7 and 8, and the 10 non-infected, anthelmintic-treated mice were drenched again each week. At week 8, 40 mice from the 260 mice infected at week 0 remained infected. All the mice in the anthelmintic-treated infected and non-infected groups and 20 of the remaining 40 infected mice were reinfected with 100 larvae at week 9, along with 10 naive mice as larvae infectivity controls. The other 20 infected mice were retained as infection controls for the dose of 400 larvae administered at week 0. All these mice were killed at 12 weeks after the first dose of 400 larvae as given, and the adult N. dub& were recovered from their intestines, sexed and counted. Mathematics. Analyses of variance, Student’s ‘t’-tests and regression analyses were done where appropriate (Snedecor & Cochran, 1968). Correlations between parasite fecundity and dose of N. dubius larvae administered were made using log lo larval dose. RESULTS Experiment 1 Similar proportions
of the inocula
of larvae were
recovered as adult worms 2 weeks after infection with 50, 100,200 or 400 larvae-90,94,94 and 95% respectively. The proportionate loss of worms was greater from infections established using 50 rather than larger doses of larvae, whereas the least loss was from the group inoculated with 400 larvae (Table 1). There were significant correlations between time after
TABLEI-INFLUENCE OF INOCULUM SIZEON ADULTNematospiroides dubius POPULATIONS IN FEMALEQUACKENBUSH MICETHROUGH TIMEAFTERINFECTION 2* Inoculum size
after infection (weeks) 4
*20 mice per group at each interval. *Mean ? S.E. of the mean; numbers of adult worms recovered.
Population dynamics of N. dubius
infection in weeks and loss of worms for all dose groups, except for mice given 400 larvae where the correlation was not satistically significant (r-values 0.98,0.83,0.91 and O-73; d.f. = 4; p < 0.001, < O-05, < 0.02 and > 0.05 for dose groups of 50, 100, 200 and 400 larvae respectively) (Table 1). Experiment 2 Ninety-two, 99, 82, 88, 86 and 92% of the larvae were recovered as adult parasites from primary infections in mice given 10, 25, 50, 100, 200 and 400 larvae respectively (Table 2). These mice lost body weight in proportion to their parasite burdens (r=O-91, d.f. =4, p < 0.01). A positive curvilinear relationship (r=0.94, d._f. =4, p < 0.01) of the form Y = a + b In X was shown between the inoculum size of larvae at primary infection and the number of adult worms recovered after secondary infection with 100 larvae, and a similar relationship held between the number of adult worms recovered after primary and the numbers recovered after secondary infection (r=0.92) (Fig. 1). The number of worms recovered after secondary infection was positively correlated with the titre of serum anti-N. dubius antibody detected at necropsy in the mice (curvilinear regression, Y=a+b In X, r=0.84; d.$=4; p < 0.05) (Fig. 2). There was an inverse relationship between the dose of larvae administered at primary infection and the fecundity of female N. dubius recovered after secondary infection (r= -0.85; d.f. =4; p < 0.05): more larvae at primary infection resulted in reduced worm fecundity at secondary infection. Likewise, an inverse relationship was noted between worm fecundity and anti-N. dubius antibody titre after secondary infection (r= -0.91; d.f. =4; p < 0.05. The female to male sex ratio of the parasite decreased after secondary infection but the differences among the groups were significant only for the comparison between the mean values from the 50 vs 400 dose groups (t = 4.09, d.f. = 50; p < 0.01). Experiment 3 There was a progressive decrease in the survival of N. dubius 3 weeks after reinfection with 100 larvae in mice which had been freed of their primary infection for increasing periods prior to reinfection (r = -0.96, d.J = 3, p < 0.01) (Fig. 3). Larval infectivity controls Similar numbers of N. dubius were recovered from the infectivity control mice given 100 larvae within and between all three experiments (range 71-84). The primary infectivity controls in Expt. 3 given 400 larvae and killed at weekly intervals from 4 to 9 and at 12 weeks after infection lost adult worms at the same rate as the mice infected with 400 larvae in Expt. 1 (87% infectivity and 0.35% loss per week). Ten naive mice infected with 100 larvae 1 week after anthelmintic treatment yielded similar numbers of worms to the untreated infectivity controls, but naive
mice treated weekly with anthelmintic for 5 weeks contained 16% fewer worms than the untreated infectivity controls (77 f 2, mean + S.E. vs 65 f 5; t = 2.2, d.J = 18, p < 0.05). Mice reinfected with 100 larvae 1 week after termination of a primary infection of 3 weeks duration with 400 larvae harboured the same numbers of N. dubius as did mice reinfected with 100 larvae 1 week after termination of a primary infection of 8 weeks duration with 400 larvae (54 + 12 vs 55 + 5). Mice which were given a concurrent infection with 100 N. dubius larvae 9 weeks after a primary infection with 400 larvae harboured 15% fewer worms 3 weeks later compared with mice infected once only with 400 larvae for 12 weeks (290 % 18 worms compared with 340 f 28). In summary, these control groups of mice showed that populations of N. dubius established from primary infection with 400 larvae decayed at the same slow rate in two experiments, that the infectivity of the parasite was similar in all inocula, that the level of protection conferred against infections 1 week after termination of the primary infection was the same at 3 and 9 weeks after primary sensitization with 400 larvae, and that any residual effects of anthelmintic on the challenge infections was not sufficient to account for the differences in worm populations between treatment groups and the other controls. DISCUSSION
Populations of N. dubius modulated their environment and increased their own survival in female Quackenbush mice. These effects were dose dependent and were evident during both primary and secondary infections. Specifically, the rate at which adult worms were lost during primary infection was retarded in proportion to the size of the primary infection. Furthermore, these effects were retained after the primary infections were removed with anthelmintic, and they enhanced the survival of N. dubius populations during secondary infection. The suppression of immunity conferred by primary infection on secondary infections with N. dubius waned as the interval between the infections was increased. Cayzer & Dobson (1983) and Behnke et al. (1983) found that adult N. dubius were responsible for the immunosuppression observed in infected mice. It has been suggested that immunosuppression may result from molecules released into the mice by N. dubius (see Mitchell, Anders, Brown, Hardman, RobertsThomson, Chapman, Forsyth, Kahl & Cruise, 1982). Indeed, cholinesterases secreted by nematodes have been implicated in similar survival strategies in various helminthoses (see Beaver & Dobson, 1978). Alternatively, antibodies that block the expression of protective immunity may be involved. The IgGl hypergammaglobulinaemia (Crandall et al., 1974) that characterises murine infection with N. dubius may be evidence for these blocking antibodies
Secondary infection* Number of mice Proportion female worms Fecundity, efd x 10-3
*Dose, 100 larvae into all mice.
Primary infections Number of mice Proportion female worms Fecundity, efd x 10-3
days before secondary infection
TABLE 2-SEXRATIO ANDFECUNDITYOF Nematospiroidesdubius RECOVERED 21 DAYS AFTERPRIMARY INFECTION OFFEMALE QUACKENBUSHMICEWITH 10-400 LARVAE, AND AT 21 DAYS AFTER SECONDARY INFECTION WITH 100 LARVAE IN THBSE MICE. Primary infections were removed with anthelmintic
Population dynamics of N. dubius
g 100 L
g g 40
s Primary infection
Fro. 1. ReIationship
between the number of adult Nematospiroides dubius recovered 3 weeks after primary infection in female Quackenbush mice and the number of adult N. dubius recovered from the same mice 3 weeks after reinfection with 100 larvae.
FIG. 2. Relationship between serum antiparasite antibody titre after reinf~tion of female Quackenbush mice with 100 Nematospiroides dubius larvae and the numbers of adult worms recovered from these mice. The mice had been in-
oculated at nrimary infection with 10 (O), 25 (e), 50 (Cl), 100 (m), 200 (A)or 400 (A)N. dubius larvae.
(Mitchell et al., 1982). Additionally, Dobson & Cayzer (1982a) found that treatment of sheathed third-stage larvae of N. dub&s with immune antiserum in v&o enhanced their survival in vivo in mice passively immunized with antiserum. The present results also showed that enhanced survival of N. dubius was directly proportional to elevated titres of serum anti-N dub&s in Q mice. This conforms with the demonstration by Dobson & Cayzer (1982b) that antiserum with high antiparasite titres from donor mice infected once only with 400 N. dubius larvae did not passively immunize recipient mice against N. dub&s as effectively as serum with lower antibody titres from other donor mice infected with 50 larvae. However, while Pritchard, Behnke &
c J:: 0 t 5 20 I 2 Q Time (weeks)
frsa of 1’ infection
FIG. 3. Decay of immunosuppression induced by a primary infection (1’) with 400 Nematospiroides dubius larvae in female Quackenbush mice measured as adult worms recovered (70) from a challenge infection of 3 weeks’ duration with 100 larvae; 1’ infections of 4-8 weeks terminated with anthelmintic 5-l weeks respectively prior to reinfection. Control groups: A-worms recovered (Vo) from naive mice infected 12 weeks previously with 400 larvae; B-worms recovered (@IO) from naive mice infected 3 weeks previously with 100 larvae as a larval infectivity control for the challenge infection; C-worms recovered (To) from mice concurrently infected for 3 weeks with 100 larvae 9 weeks after a 1o infection with 400 larvae. Means for 10-20 mice plus 2 S.E.S of the mean are shown.
Williams (1984) detected binding of murine antibodies to adult N. dub&s in serum from primary infections with 400 larvae in mice, they were unable to differentiate how these could block the action of protective responses to N. dubius. Residual levels of immunosuppression were detected following challenge infection in mice for at least 5 weeks after the primary infection that induced the effect was terminated, but it decreased with time and by extrapolation (Fig. 3) Q mice may have shown complete protection to reinfection by 7 weeks after the primary infection had been terminated. However, the control data from Expt. 3 also showed that suppression, evident following primary infection with 400 larvae, did not completely override a protective reaction to a concurrent challenge infection with 100 larvae which appeared as a partial ‘self-cure’. Classical ‘self-cure’ reactions exhibiting the expulsion of whole populations of adult worms, as described for ovine haemonchosis (Stall, 1929), do not result at challenge infection with N. dubius in all strains of mice (Cypess & Zidian, 1975). However, Cypess and van Zandt (1973) demonstrated that repeated administration of N. dubius larvae to outbred S-W mice generated the rapid expulsion of established aduh worms. The duration of suppression may be the time required for the catabolism of blocking antibodies, which may be more rapid than that of protective antibodies. Pritchard, Williams, Behnke & Lee (1983) demonstrated that protective antibodies against both L4 and adult N. dubius reside in IgGl, levels of
COLINDOBSON,PETRUSSITEPUand PAUL J. BRINDLEY
which were elevated tenfold in infected mice, whereas they did not find protective antibodies in IgG2a, IgGZb, IgM or IgA. Furthermore, they showed that levels of these latter immunoglobulin isotypes were depressed or unaffected by infection with N. dubius. Blocking antibodies may reside in any of these immunoglobulins, including IgGl where up to 48% may be specific for N. dubius antigens (Pritchard et al., 1983). Blocking antibodies in Q mice after primary infection would have been catabolized to extinction by 7 weeks after the primary infection had been removed. The half-life of IgGl extends beyond those of the other murine immunoglobulins (Spiegelberg, 1974) and elevated titres of IgGl enhance the catabolism of other IgG isotypes (Fahey & Sell, 1965). Female Q mice lost body weight in proportion to the number of N. dubius that they harboured. N. dubius is pathogenic in mice (Spurlock, 1943; Baker, 1955; Liu, Cypess & van Zandt, 1974; Mitchell & Prowse, 1979) and the duration of enhanced parasite survival from a challenge infection may equate with the time required to repair parasite induced damage rather than with the decay of blocking antibodies or other mediators that suppressed protective immunity. Furthermore, Hagan and Wakelin (1982) showed that N. dubius inhibited the migration of lymphoblasts to the intestines of infected mice. If this influence was proportional to size of the infection it could in turn enhance the survival of N. dubius challenge infections. thank Mrs M. E. Owen for fine technical assistance. This work was supported by the
Australian Research Grants Scheme.
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